Allergen-induced late-phase reactions (LPRs) have been implicated in the pathogenesis of a variety of allergic diseases. Histologically, the LPRs are characterized by various types of cellular infiltrates, including T cells. The molecular and functional basis of T-cell infiltration remain undefined. We postulate that, in the LPRs, T cells are recruited and activated locally in an AG-specific manner, and are directly involved in the regulation of allergic inflammation. The aim is to provide evidence for the Ag specificity and functional importance of the infiltrating T cells in the LPRs. The model for these studies is specific T-cell responses to a well-defined ragweed allergen, Amb a V, in human atopic subjects. We will combine molecular and cellular approaches to characterize the Ag- specific recruitment and activation of the infiltrating T cells in the skin chamber blister, lung bronchoalveolar lavage (BAL), and the respective biopsy samples. First, the specific response of T cells to Amb a V will be determined, and the Amb a V-specific T-cell clones will be generated from the peripheral blood, the skin and lung samples following challenge with Amb a V. Second, to obtain direct evidence for the recruitment of specific T cells, "gene-targeting" experiments will be performed, in which the Amb a V-specific TcR alpha and beta genes of selected peripheral blood T-cell clones will serve as "trace markers" and will be targeted" in the skin and lung samples by employing the polymerase chain reaction (PCR) technique and DNA sequencing. The relative frequency of specific T cells will be determined by quantitative PCR, using modified Amb a V-specific TcR cRNAs as internal standards. Experiments will involve antigen-specific human T-cell clones and lines, including quantitation of cytokine-gene expression and production, as well as sequencing of TcR genes. Based on TcR sequence similarities in the T-cell clones generated from peripheral blood, we will design specific oligonucleotides to investigate the relationships of specific TcR gene sequences in the LPRs to Amb a V. The accomplishment of these goals will provide a novel model for investigating the LPR, and its underlying immunoregulatory mechanisms at the molecular and cellular level.